CN115785237B - Recombinant botulinum toxin and preparation method thereof - Google Patents
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Abstract
The invention relates to the technical field of biology, in particular to a recombinant botulinum toxin and a preparation method thereof. According to the invention, through the design optimization of heavy chain and light chain sequences of the recombinant botulinum toxin, the introduction of amino acid residues which are not present in the sequence of the botulinum toxin itself due to the addition and cleavage of protein tags is realized, and meanwhile, the efficient and stable expression of the botulinum toxin and the preparation of the active botulinum toxin can be performed, and the purification process of toxin proteins can be greatly simplified, so that the efficient preparation of the active botulinum toxin is realized. The preparation method of the botulinum toxin provided by the invention can be used for preparing the botulinum toxin rapidly and efficiently, is easy for large-scale mass production, and greatly reduces the production cost of the botulinum toxin.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a recombinant botulinum toxin and a preparation method thereof.
Background
Botulinum toxin is a neurotoxin secreted by a clostridium botulinum bacterium and can be divided into a total of 7 serotypes a-G based on antigenicity. Botulinum toxin is strongly neurotoxic and can cause neuroparalysis at very small doses, and when a human ingests food containing botulinum toxin, the latency period is 6h to 12d, and clinical symptoms generally appear in 3 to 4d, and the patient finally dies due to respiratory failure. Botulinum toxin is very toxic, equivalent to ten thousand times that of potassium cyanide.
Clostridium botulinum itself produces a toxin protein complex of molecular weight 900kD, consisting of three parts, hemagglutinin protein (HA), non-toxin non-hemagglutinin protein (NTNH) and neurotoxic protein. The molecular weight of the neurotoxic protein is 150kD, the neurotoxic protein is produced by clostridium botulinum under anaerobic condition, and then the neurotoxic protein is digested by protease produced by clostridium botulinum to form light peptide and heavy peptide, wherein the light chain is close to the N end and is a catalytic domain, and the heavy chain is close to the C end and is a receptor binding domain. The heavy chain and the light chain are connected by disulfide bonds to form the toxic botulinum toxin. After humans eat food containing botulinum toxin by mistake, proteases in the digestive tract cannot degrade the food due to the presence of the protective protein, so that the active ingredient enters the blood through the intestinal wall; thereafter, through blood circulation, it binds to the receptor site of motor or sympathetic nerve cells, with the heavy chain binding to the receptor; botulinum toxin enters nerve cells by endocytosis of the nerve cells, and because a cytoplasmatic matrix presents a reductive environment, double bonds between a heavy chain and a light chain are reduced and opened, the light chain is released from a complex, and the snap-25 protein of a postsynaptic membrane is cleaved, so that synthesis of SNARE complex protein is prevented, release of acetylcholine is inhibited, serious paralysis symptoms of muscles are caused, and further respiration is inhibited, and death is caused.
Because of its neuroparalysis, botulinum toxin was initially used to treat facial muscle spasms and other muscle movement disorders, and the botulinum toxin was used to paralyze the muscle nerves in order to stop the muscle spasms. Botulinum toxin is also used in medical cosmetology to relax excessively contracted small muscles by blocking nerve impulses between nerves and muscles, thereby achieving a wrinkle-removing effect; or the characteristics of the muscle can be temporarily paralyzed to make the muscle shrink due to the loss of functions so as to achieve the purpose of sculpturing the body lines. In addition, botulinum toxin has effects of inhibiting limb cramps and anti-depression.
The existing extraction method of the botulinum toxin mainly comprises the steps of expressing proteins through anaerobic fermentation of clostridium botulinum, and purifying the proteins from a culture solution through multistage purification, namely, dialysis and a series of acid precipitation, wherein the method takes about 5 days from fermentation to final obtaining of the botulinum toxin proteins. The effective component obtained by the method is a 900kD botulinum toxin complex, and a part of lectin protein of the botulinum toxin is further removed by an ion exchange method to obtain a toxin complex of about 450 kD; and removing other proteins by changing the pH to obtain the botulinum toxin with 150 kD.
In situ extraction of botulinum toxin by Clostridium botulinum is a relatively traditional method, which is well established in theory. However, fermentation of clostridium botulinum requires a very strict anaerobic environment and is very temperature sensitive. Therefore, special fermentation equipment is required to perform large-scale production. In addition, since the expressed botulinum toxin does not carry an affinity chromatography tag, multiple purification steps are required in the extraction to obtain a relatively pure protein. Moreover, the longer the preparation process, the greater the probability of accidental contamination during the process due to the strong toxicity of botulinum toxin.
In addition to methods for in situ extraction of botulinum toxin by Clostridium botulinum, other chassis cells may be used to heterologously express recombinant botulinum toxin. Recombinant botulinum toxins are currently expressed in substantially all manner of cells of the chassis using E.coli. The method for preparing the botulinum toxin by the escherichia coli mainly comprises the following steps: the plasmid for expressing botulinum toxin is transferred into E.coli using plasmid transfection techniques and the 150kD botulinum neurotoxin is induced using an inducer (e.g., IPTG).
Existing recombinant expression of botulinum toxin is divided into two modes, single-stranded and double-stranded expression. Double-stranded expression was designed according to the existing protein structure (protein data bank id:3V 0C). In this structure, there is a distinct interaction site between the heavy and light chains. By co-expression, a botulinum toxin light-heavy chain complex can be obtained, and because of the cysteine residues at the carboxy terminus of the light chain and the amino terminus of the heavy chain, disulfide bonds can be formed between the light chain and the heavy chain, thereby forming an active protein complex. There is also a method of recombinant expression of single-chain botulinum toxin, but since Escherichia coli lacks necessary protease and cannot digest the full-length single-chain protein, it is necessary to introduce protease cleavage sites between the light chain and the heavy chain exogenously and activate the protein by in vitro cleavage. The activated protein has similar activity to commercial botulinum toxin products.
Although the recombinant expression of botulinum toxin by E.coli has the advantages of high speed and the like, in order to avoid introducing amino acid residues which are not present in the sequence of the botulinum toxin, protein without an affinity tag is expressed in the prior art, the purification method still mainly comprises ammonium sulfate precipitation, the process is complicated, and the botulinum toxin has high toxicity and still has the possibility of causing accidental pollution. Single chain fusion expression, however, entails the introduction of protease cleavage sites between the heavy and light chains, as well as additional amino acid residues. While the introduction of additional sequences may introduce unknown antigenicity, may elicit an unknown immune response after injection into the body, and may potentially adversely affect the body, no related products are currently approved for use, despite the fact that recombinant botulinum toxin has comparable activity to natural toxins.
Disclosure of Invention
The invention aims to provide a recombinant botulinum toxin and a preparation method thereof.
The invention aims to prepare the botulinum toxin protein with natural sequence and biological activity and realize efficient and stable expression of the protein in a biological chassis. For this purpose, the sequences of the heavy and light chains of the recombinant botulinum toxin are first specifically designed and optimized. In order to simplify the subsequent purification process and avoid introducing amino acid residues which are not present in the sequence of the botulinum toxin itself as much as possible, the invention adopts a double-stranded expression mode and introduces a protein tag. According to the invention, a great deal of researches show that in the process of double-chain expression of recombinant botulinum toxin, the expression quantity of the light chain is far higher than that of the heavy chain, so that the protein tag is added on the heavy chain, and the interference of redundant light chain on purification is reduced. During the introduction of protein tags and removal of tags, the introduction of amino acid residues that are not present in the sequence of the botulinum toxin itself is typically caused. The invention avoids the introduction of extra amino acid residues through sequence design.
Specifically, the invention provides the following technical scheme:
in a first aspect, the present invention provides a recombinant botulinum toxin comprising a heavy chain and a light chain linked by disulfide bonds; the amino acid sequence of the heavy chain sequentially comprises a protein tag sequence, an ENLYFQ polypeptide sequence and a heavy chain sequence of the botulinum toxin from the N end to the C end, wherein the 1 st amino acid of the N end of the heavy chain sequence of the botulinum toxin is glycine.
The protein tag introduced into the heavy chain sequence of the recombinant botulinum toxin can be any protein tag which facilitates protein purification, including but not limited to His-tag, gst, MBP, strep, flag and the like.
In some embodiments of the present invention, the protein tag is exemplified by His-tag, but is not limited to this in practical application.
The heavy chain sequence of the botulinum toxin can be the complete heavy chain sequence of the botulinum toxin or a part of the heavy chain sequence is intercepted, but the 1 st amino acid at the N end of the sequence needs to be ensured to be glycine, and the botulinum toxin has the activity after being connected with the light chain through disulfide bonds.
The heavy chain composition structure of the recombinant botulinum toxin provided by the invention is applicable to various serotypes of botulinum toxin (such as A type, E type and the like).
Specifically, for botulinum toxin A, the heavy chain is taken from the 445 th glycine of the full-length single chain of botulinum toxin A to the C-terminal, the N-terminal 6 amino acid residues of the cleavage sequence (ENLYFQ ∈G, arrow mark is the cleavage position) of TEV protease are introduced at the N-terminal of the 445 th glycine, and the protein tag sequence is introduced at the N-terminal 6 amino acid residues of the cleavage sequence of TEV protease. The sequence design ensures that the heavy chain is subjected to TEV enzyme digestion, no amino acid residues with the sequence which does not exist (namely, no artificially introduced protein tag sequence and enzyme digestion site sequence are remained, and the heavy chain of the botulinum toxin still maintains the original sequence), and the digested heavy chain has the biological activity of the natural botulinum toxin after being linked with the light chain through disulfide bonds.
In some embodiments of the invention, the heavy chain sequence of the botulinum toxin described above is the amino acid sequence from position 445 to position 1296 of botulinum toxin a.
Among the amino acid sequences 445 to 1296 of botulinum toxin A described above, amino acid 445 is glycine. The amino acid sequence from position 445 to position 1296 of botulinum toxin A may be the sequence of native botulinum toxin A (e.g., from position 445 to position 1296 of the sequence shown in SEQ ID NO. 1) or may be a mutated botulinum toxin A sequence, which still requires that the amino acid at position 445 be glycine and be capable of linking to the light chain to form a biologically active botulinum toxin.
In some embodiments of the invention, the amino acid sequences 445 to 1296 of botulinum toxin A described above are based on the botulinum toxin A sequence shown in SEQ ID NO.1 (the botulinum toxin encoding gene sequence shown in SEQ ID NO.1 is shown in SEQ ID NO.5, the sequence shown in SEQ ID NO.5 is a sequence optimized for expression in E.coli). The amino acid sequences 445 to 1296 of botulinum toxin A may also be variants of the above amino acid sequences, provided that the 445 amino acid is glycine and is capable of linking with the light chain to form a biologically active botulinum toxin.
The recombinant botulinum toxin can be subjected to TEV protease digestion to generate the botulinum toxin with the protein tag sequence completely removed, and amino acid residues which are not existed in the sequence of the botulinum toxin can not be remained.
Preferably, the amino acid sequence of the heavy chain of the recombinant botulinum toxin further comprises a methionine in the direction from the N-terminus to the C-terminus and an amino acid sequence consisting of 1 to 3 amino acid residues other than cysteine at the N-terminus of the protein tag sequence;
in some embodiments of the invention, the amino acid sequence of the heavy chain is, in order from the N-terminus to the C-terminus: m-an amino acid sequence consisting of 1 to 3 amino acid residues except cysteine-a protein tag sequence-ENLYFQ-botulinum toxin heavy chain sequence (glycine G at position 1 of N-terminal).
In some embodiments of the invention, the amino acid sequence of the heavy chain is, in order from the N-terminus to the C-terminus: m-amino acid sequence consisting of 1 to 3 amino acid residues other than cysteine-protein tag sequence-amino acids 445 to 1296 of ENLYFQ-botulinum toxin A.
In some embodiments of the invention, the amino acid sequence of the heavy chain is, in order from the N-terminus to the C-terminus: MK-protein tag sequence-ENLYFQ-amino acids 445 to 1296 of botulinum toxin A.
In some embodiments of the invention, the amino acid sequence of the heavy chain is shown in SEQ ID NO. 2.
The heavy chain amino acid sequence of the recombinant botulinum toxin is a sequence carrying a protein tag, the protein tag can be cut off by TEV protease enzyme, the heavy chain without the tag and the residual amino acid residue at the enzyme cutting site is produced, and antigenicity except the botulinum toxin is not produced.
Based on the heavy chain sequence, the invention tries to select and optimize the light chain sequence, and determines the position from the N end of the full-length single chain of the botulinum toxin to the 438 th lysine, so that the biological activity of the light chain can be well ensured by taking the heavy chain sequence as the light chain sequence. The light chain and the heavy chain designed above can form disulfide bonds with high efficiency, and the botulinum toxin with biological activity without introducing any amino acid residue which does not exist in the sequence of the botulinum toxin is generated after the light chain and the heavy chain are subjected to the enzyme digestion of TEV protease.
Preferably, in the recombinant botulinum toxin, the amino acid sequence of the light chain is from position 1 to position 438 of botulinum toxin a.
The amino acid sequences from position 1 to 438 of botulinum toxin A may be the sequence of native botulinum toxin A (e.g., positions 1 to 438 of the sequence shown in SEQ ID NO. 1), or may be the sequence of mutant botulinum toxin A, which is capable of forming a biologically active botulinum toxin by ligation to the heavy chain.
In some embodiments of the invention, the amino acid sequence of the light chain is shown at positions 1 to 438 of the sequence shown in SEQ ID NO. 1.
The heavy and light chains described above retain the original cysteines at positions corresponding to the amino acids 430, 454 of the full length single chain of botulinum toxin A, so disulfide bonds may be formed between the light and heavy chains, and efficient disulfide bond formation is facilitated by the light and heavy chains of the design described above.
In a second aspect, the invention provides a nucleic acid molecule encoding a recombinant botulinum toxin as described above.
Based on the amino acid sequences of the heavy and light chains of the recombinant botulinum toxins described above, one skilled in the art can obtain nucleotide sequences encoding the nucleic acid molecules of the heavy and light chains described above. The nucleotide sequence of the above-described nucleic acid molecule is not unique based on the degeneracy of the codons, and all nucleic acid molecules capable of encoding the heavy and light chains are within the scope of the invention.
In some embodiments of the invention, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain is shown in SEQ ID NO. 3.
In some embodiments of the invention, the nucleotide sequence of the nucleic acid molecule encoding the light chain is set forth in SEQ ID NO. 4.
The nucleic acid molecule can realize the high-efficiency stable expression of the light chain and the heavy chain in escherichia coli and vibrio natrii.
In a third aspect, the invention provides a biological material comprising a nucleic acid molecule as described above; the biological material is an expression cassette, a vector or a host cell.
In some embodiments of the invention, the expression cassette comprising the nucleic acid molecule is operably linked to a promoter.
Other transcription and translation regulatory elements such as terminators, enhancers and the like can also be included in the expression cassette according to the expression needs and the difference of the upstream and downstream sequences of the expression cassette.
In some embodiments of the invention, the vectors containing the nucleic acid molecules are plasmid vectors, including replicative vectors and non-replicative vectors. The vector containing the nucleic acid molecule is not limited to a plasmid vector, and may be a vector such as phage or virus.
In some embodiments of the invention, the plasmid vector contains a nucleic acid molecule encoding the heavy chain described above and/or a nucleic acid molecule encoding the light chain described above.
In some embodiments of the present invention, the plasmid vector is a pqink-based backbone vector into which a nucleic acid molecule encoding the heavy chain and a nucleic acid molecule encoding the light chain are linked in series. The nucleic acid molecule encoding the heavy chain and the nucleic acid molecule encoding the light chain each comprise a corresponding protein expression gene and each possess an independent promoter, regulatory factor, ribosome binding site and terminator.
In some embodiments of the present invention, the host cell is E.coli or Vibrio natrii, but the kind of host cell is not limited thereto, and may be any microbial cell or animal cell that can be used for protein expression.
In a fourth aspect, the present invention provides an engineered bacterium for the production of recombinant botulinum toxin, the engineered bacterium comprising a nucleic acid molecule as described above.
In some embodiments of the invention, the engineered bacterium is escherichia coli or vibrio natrii.
In the invention, both Escherichia coli and Vibrio natrii can be used as chassis bacteria for expressing recombinant botulinum toxin. However, the present invention surprisingly found that Vibrio natrii is more effective in promoting disulfide bond formation between the light chain and the heavy chain of botulinum toxin, and thus enables faster and better successful synthesis of botulinum toxin having biological activity.
Moreover, compared with the method that escherichia coli is easy to be infected by phage to generate cell rupture, content is dissolved out, and the preparation of botulinum toxin by taking vibrio natriuretic as chassis bacteria can also reduce the risk of phage infection, effectively reduce the risk of environmental pollution caused by toxic proteins, and shorten the cell culture period. In some embodiments of the invention, the engineered bacterium contains a plasmid vector carrying the nucleic acid molecule described above.
In some embodiments of the invention, the engineering bacteria are obtained by introducing a plasmid vector carrying the nucleic acid molecule described above into vibrio natrii. Wherein the nucleic acid molecule encoding the heavy chain and the nucleic acid molecule encoding the light chain may be on the same plasmid vector or may be on separate plasmid vectors.
In some embodiments of the invention, the engineering bacterium is obtained by introducing a plasmid vector carrying the nucleic acid molecule into escherichia coli. Wherein the nucleic acid molecule encoding the heavy chain and the nucleic acid molecule encoding the light chain may be on the same plasmid vector or may be on separate plasmid vectors.
In a fifth aspect, the present invention provides the use of a recombinant botulinum toxin or the nucleic acid molecule or the biological material or the recombinant vibrio natrii is described above in the preparation of a botulinum toxin.
In some embodiments of the invention, the above-described application includes: modifying the host cell so that the host cell expresses the recombinant botulinum toxin described above or contains the nucleic acid molecule, culturing the host cell, collecting the recombinant botulinum toxin expressed thereby, purifying by affinity chromatography and performing TEV protease cleavage to obtain the protein-tagged botulinum toxin.
In some embodiments of the invention, the above-described application includes: culturing the recombinant vibrio natrii, collecting the expressed recombinant botulinum toxin, purifying by affinity chromatography and cutting by TEV protease to obtain the protein-tag-removed botulinum toxin.
In a sixth aspect, the present invention provides a method of preparing a botulinum toxin, the method comprising: modifying the host cell such that the host cell expresses the recombinant botulinum toxin described above, culturing the host cell, purifying the recombinant botulinum toxin from the culture using affinity chromatography, and subjecting the purified recombinant botulinum toxin to TEV protease cleavage.
In the above method, the column used for the affinity chromatography may be selected according to the protein tag contained in the heavy chain.
In some embodiments of the invention, his-tag affinity chromatography columns are used corresponding to the His-tag protein tags employed. In practical application, the corresponding purification method can be selected and used according to different protein tag sequences.
In the above method, the host cells are cultured to obtain a culture, and the culture is subjected to affinity chromatography purification by separating the supernatant after lysing the cells.
In some embodiments of the invention, the method further comprises the step of sequentially performing anion exchange chromatography and molecular sieve chromatography purification after the TEV protease cleavage.
In the above purification, anion exchange chromatography may be performed using an anion exchange column (e.g., source Q). Molecular sieve chromatography can be performed by using molecular sieve columns such as SuperDex 200.
The purification method can remarkably improve the purification efficiency and purity of the recombinant botulinum toxin.
In the method, the whole purification process can be completed in less than one day, compared with the complicated purification process of the label-free protein, the purification process is greatly simplified, the manpower and materials are saved, and the possibility of accidental pollution to the environment such as instruments is effectively reduced.
For host cells, in some embodiments of the invention, the host is Vibrio natrii.
The present invention has unexpectedly found that Vibrio natrii is able to more efficiently promote disulfide bond formation between the light chain and the heavy chain of botulinum toxin, thereby enabling faster and better successful synthesis of botulinum toxin having biological activity.
Moreover, compared with the method that escherichia coli is easy to be infected by phage to generate cell rupture, content is dissolved out, and the preparation of botulinum toxin by taking vibrio natriuretic as chassis bacteria can also reduce the risk of phage infection, effectively reduce the risk of environmental pollution caused by toxic proteins, and shorten the cell culture period.
The invention has the beneficial effects that: according to the invention, through the design optimization of heavy chain and light chain sequences of the recombinant botulinum toxin, amino acid residues which are not existed in the sequence of the botulinum toxin are not introduced by adding and cutting protein tags, and meanwhile, the efficient and stable expression of the botulinum toxin and the preparation of the active botulinum toxin can be carried out, and the purification process of toxin proteins can be greatly simplified, so that the efficient preparation of the active botulinum toxin is realized.
The preparation method of the botulinum toxin provided by the invention can be used for rapidly and efficiently preparing the botulinum toxin, has the advantages of short host culture period, difficult phage pollution, simple and feasible toxin protein purification process, easiness in large-scale mass production, great reduction of the preparation cost of the botulinum toxin, expected bioactivity of the prepared botulinum toxin, and good application prospect compared with the natural botulinum toxin.
Drawings
In order to more clearly illustrate the invention or the technical solutions in the prior art, the drawings that are used in the description of the embodiments or the prior art will be briefly described one by one, it being obvious that the drawings in the description below are some embodiments of the invention, and that other drawings can be obtained from these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows PCR bands of the products P1, P2, P3 in example 1 according to the invention.
FIG. 2 shows the PCR screening bands of example 2 of the present invention, wherein 2,4 are expected.
FIG. 3 is a construction diagram of a tandem plasmid in example 2 of the present invention.
FIG. 4 shows the results of His-tag affinity chromatography purification in example 5 of the present invention.
FIG. 5 shows the result of anion exchange column purification in example 5 of the present invention.
FIG. 6 shows the result of SDS-Page verification of the purification of the anion exchange column in example 5 of the present invention, FT is the flow-through liquid, and numbers 7 to 14 are the numbers of the collection tubes of the anion exchange column in FIG. 5, respectively.
FIG. 7 shows the result of purifying SUPERDEX200 as a molecular sieve in example 5 of the present invention.
FIG. 8 shows the result of SDS-PAGE verification of the molecular sieve SUPERDEX200 purification in example 5 of the present invention, wherein the numbers 11 to 18 are the numbers of the collection tubes of the molecular sieve SUPERDEX200 in FIG. 7, respectively.
FIG. 9 shows the result of SDS-PAGE verification of molecular sieve SUPERDEX200 in example 6 of the present invention.
FIG. 10 is a graph showing the concentration of botulinum toxin protein in example 7 of the present invention.
FIG. 11 shows the results of the gastrocnemius injection experiment for mice in example 7 of the present invention after 12 hours.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
EXAMPLE 1 construction of a botulinum light chain heavy chain expression vector
The PQlink expression vector (http:// www.youbio.cn/product/vt 8126) was purchased from Ubbelopsis grossedentata, designed based on the Clostridium botulinum expressed botulinum protein sequence (UniProtKB/Swiss-Prot: P0DPI0.1) published by NCBI (national center for biological information, website: https:// www.ncbi.nlm.nih.gov /), and was delegated to the synthesis of the botulinum toxin encoding gene by Kirschner Biotech Co., ltd.
A PCR amplification method is adopted, PQLink homology arms are respectively introduced at the upstream and downstream of the light chain and the heavy chain, and His tag and TEV enzyme cutting sites are introduced at the upstream of the heavy chain. Using the KOD Mix system, 20. Mu.L of each system reaction was performed. The amplification procedure was: denaturation at 95℃for 15 s; annealing at 56℃for 10s and extension at 68℃for 30s, the whole reaction was 34 cycles. The reaction fragments were collected by nucleic acid gel electrophoresis and designated as P1 and P2. And the corresponding primer of PQlink is used for carrying out PCR amplification on the vector, linearizing the vector, collecting a reaction fragment by using nucleic acid gel electrophoresis, marking the reaction fragment as P3, and the reaction system is 50 mu L, wherein the amplification procedure is as follows: denaturation at 95℃for 15 s; annealing at 56℃for 10s and extension at 68℃for 60s, the whole reaction was 34 cycles. As a result, as shown in FIG. 1, the P1 band size was about 1200bp, the P2 band size was about 2600bp, and the P3 band size was about 5000 bp. The sizes are all in line with expectations. The primers used are as follows.
Primer BotLc-5:5' -GAGGAGAAATTAACTATGCCGTTCGTTAACAAACAGTTC-3’
Primer BotLc-3:5' -GTCCTAGGCGGCCGCTTATTTAGAGGTGATGATACCACG-3’
Primer BotHc-5:
5’-GAGGAGAAATTAACTATGAAACATCACCATCACCATCACGAGAATCTTTATTTTCAGGGTTACAACAAAGCTCTGAACGA-3’
primer BotHc-3:5' -GTCCTAGGCGGCCGCTTACAGCGGACGTTCACCCCAACCGT-3' (underlined to indicate PQlink homology arms)
Primer PQlink-5v:5'-TAAGCGGCCGCCTAGGACCCAGC-3'
Primer PQlink-3v:5'-CATAGTTAATTTCTCCTCTTTAA-3'.
And cutting the PCR reaction strip into gel, and recovering by using a TIANGEN gel recovery kit, wherein the operation steps are the same as the instruction book. The light and heavy chain recovery fragments P1 and P2 are subjected to homologous recombination with P3 respectively, and the kit selects a noniprandial homologous recombination kit (ClonExpress II One Step Cloning Kit), and the operation steps are as described in the specification.
10 μl of the reaction product was taken and TOP10 competent (commercially available) was added, and the mixture was heat-shocked at 42℃for 90s; 200. Mu.L of fresh LB medium was added for 30min, and then plated on a resistant LB solid plate (containing 50. Mu.g/mL ampicillin) and incubated overnight at 37 ℃. The transformant is picked up for expansion culture, then plasmid is extracted, PCR (primer selection light and heavy chain corresponding primers) and sequencing verification are sequentially carried out, sequencing company is Bo Rui Ke Biotechnology Co., ltd, and sequencing primer is company universal primer PQlink-F, PQlink-R.
Primer PQlink-F:5'-TATAAAAATAGGCGTATCACGAGG-3'
Primer PQlink-R:5'-CCAGTGATTTTTTTCTCCATTTT-3'.
The coding gene sequence of the heavy chain of the botulinum toxin obtained by PCR amplification is shown as SEQ ID NO.3, the amino acid sequence of the heavy chain is shown as SEQ ID NO.2, the coding gene sequence of the light chain is shown as SEQ ID NO.4, and the amino acid sequence of the light chain is shown as positions 1-438 of SEQ ID NO. 1.
EXAMPLE 2 construction of botulinum light chain heavy chain tandem Co-expression vectors
Plasmids in which the light chain and the heavy chain were expressed separately were obtained in example 1, and were designated as B1 and B2 in this order.
B2 was subjected to SwaI cleavage (both SwaI and buffer3.1 were purchased from NEB) in the following manner: mu.L of plasmid, 0.5. Mu.L of enzyme, 1. Mu.L of Buffer3.1, and water was made up to 10. Mu.L. And (5) enzyme cutting for 2 hours at room temperature. The system was then placed at 65℃for 30min, the product being designated P4.
PCR reaction is carried out by taking B1 as a template, the reaction system is 20 mu L, and the amplification procedure is as follows: denaturation at 95℃for 15 s; annealing at 56℃for 10s and extension at 68℃for 30s, the whole reaction was 34 cycles. The primers were as follows:
primer PQlink-5ter:5'-TAACAACACCATTTGTCGAGAA-3';
primer PQlink-3ter:5'-CCATTTGTAACCACTCCATTT-3'.
And (3) collecting reaction fragments by using nucleic acid gel electrophoresis, cutting a PCR reaction strip into gel, and recovering by using a TIANGEN gel recovery kit, wherein the operation steps are the same as the instruction book, and the product is marked as P5. The recovered fragments P4 and P5 were subjected to homologous recombination, and the kit was selected from the nonizane homologous recombination kit (ClonExpress II One Step Cloning Kit), and the procedure is as described in the specification.
10 μl of the reaction product was taken and TOP10 competent (commercially available) was added, and the mixture was heat-shocked at 42℃for 90s; 200. Mu.L of fresh LB medium was added for 30min, and then plated on a resistant LB solid plate (containing 50. Mu.g/mL ampicillin) and incubated overnight at 37 ℃. The transformants were picked for expansion, plasmids were extracted, and PCR (primer selection BotHc-5, botLc-3) and sequencing verification were performed sequentially. As shown in FIG. 2, lanes 2 and 4 are about 4000bp, the size accords with the expectation, the corresponding strain is selected for sequencing, the sequencing company is Bocinia biotechnology Co., ltd, and the sequencing primers are company universal primers PQlink-F and PQlink-R.
Primer PQlink-F:5'-TATAAAAATAGGCGTATCACGAGG-3';
primer PQlink-R:5'-CCAGTGATTTTTTTCTCCATTTT-3'.
The results showed that the sequencing results were correct. The schematic diagram of the constructed plasmid is shown in FIG. 3, wherein Bonta HC is a heavy chain of botulinum toxin with an N-terminal His tag, bonta LC is a light chain of botulinum toxin, promoters of heavy chain and light chain genes are t5 promoters, regulatory elements are lac operator, and terminators are lambda t0 terminator.
EXAMPLE 3 expression of recombinant botulinum toxin Using E.coli
2. Mu.L of the co-expression plasmid prepared in example 2 was added to Origami DE3 competence (commercially available), and heat-shocked at 42℃for 90s; 200. Mu.L of fresh LB medium was added for 30min, and then plated on a resistant LB solid plate (per liter of composition, 10g of NaCl,10 g of tryptone, 5g of yeast extract, 10g of agar, 50. Mu.g/mL of ampicillin) and cultured overnight at 37 ℃.
The monoclonal transformation was inoculated in 10mL of LB medium (per liter of the composition, 10g of NaCl,10 g of tryptone, 5g of yeast extract, 50. Mu.g/mL of ampicillin) and cultured overnight at 37℃in a shaker at 220 rpm. Then, the bacterial solution was inoculated into 1L of LB medium (containing 50. Mu.g/mL ampicillin) and cultured at 220rpm and 37 ℃. Culturing for 6 hours, OD is 1.5, cooling to 18 ℃, adding 500mM IPTG and inducing with 400 mu L.
EXAMPLE 4 expression of recombinant botulinum toxin Using Vibrio natrii
Vibrio natrii competence is purchased at synthetic genomics under the trade name Vmax TM Express. 2. Mu.L of the co-expression plasmid prepared in example 2 was added to the vibrio natriuretic competent cells, and the mixture was subjected to electric shock transformation at 1500V for 5ms. Electric shock conversion cups were purchased from biorad under the trade designation 165-2086 2mm. The electric shock transformation product was added to 1mL of high salt LB medium (30 g of NaCl,10 g of tryptone, 5g of yeast extract per liter of ingredients). Shaking culture was carried out at 37℃for 1 hour. Coated onto a high-salt LB plate (30 g of NaCl,10 g of tryptone, 5g of yeast extract, 10g of agar, 50. Mu.g/mL ampicillin per liter).
The monoclonal transformation was inoculated in 10mL of high-salt LB medium (30 g of NaCl,10 g of tryptone, 5g of yeast extract, 50. Mu.g/mL of ampicillin per liter), and cultured overnight at 37℃in a shaker at 220 rpm. The bacterial solution was inoculated into 1L of a high-salt LB medium (30 g of NaCl,10 g of tryptone, 5g of yeast extract, 50. Mu.g/mL of ampicillin per liter) and cultured at 220rpm at 37 ℃. Culturing for 3 hours, OD is 1.5, cooling to 18 ℃, adding 500mM IPTG and inducing with 400 mu L.
EXAMPLE 5 purification of botulinum toxin
For the recombinant botulinum toxins expressed in examples 3 and 4, the botulinum toxin proteins expressed by Vibrio natriuretic Vmax and E.coli Origami were purified simultaneously under the same conditions as follows:
bacterial liquid was collected and centrifuged at 3800rpm for ten minutes in a low-speed high-capacity centrifuge. The supernatant was removed and each liter of bacterial entity was resuspended with 25mL of lysis buffer (25 mM Tris 8.0, 150mM NaCl).
Ultrasonic cracking, conditions: horn 10, power 350W, ultrasound for 2s, stop for 2s, 4min per liter of fungus entity (e.g. 6L of fungus collected, ultrasound time should be 24 min), ultrasound to clarified the fungus fluid.
A50 mL centrifuge tube was used as a container, and centrifuged at 13000rpm at high speed for 1 hour. Taking the supernatant.
The supernatant was centrifuged at high speed and then passed through His-tag column with 3mL of column per liter of fungus bodies. Twice.
Wash buffer (25mM Tris 8.0, 150mM NaCl,10mM imidazole), 50mL per liter of bacterial entity.
Elute buffer (25mM Tris 8.0, 50mM NaCl,250mM imidazole), 10mL per liter of bacterial entity was washed and the eluate was collected. 10mL of the eluate was subjected to digestion with 100U of TEV enzyme (available from Biyun Tian product No. P2307).
Electrophoresis detection, 5 Xnon-denatured, non-reduced protein loading buffer was purchased from YeasenChina under accession number 20317ES, methods of preparation are described in the specification. Wherein, the centrifugal precipitation, the supernatant loading amount of the centrifugal precipitation is 2 mu L, the high imidazole eluent loading amount is 5 mu L, and the polyacrylamide gel electrophoresis is carried out. The detection results are shown in FIG. 4.
Comparing the results of expressing recombinant botulinum toxin by Vibrio natrii with that of the E.coli origin, the presence of two mutations in the E.coli origin cytoplasmic disulfide reduction pathway at trxB/gor can increase disulfide bond formation in the E.coli cytoplasm. Unexpectedly, in vibrio natrii, normal disulfide bond can be formed between heavy chain and light chain of botulinum toxin, and the purity of protein expressed by vibrio natrii is similar to Origami expression result. In FIG. 4, it is shown that during the high imidazole elution, the purity of the bands of Vibrio natriuretic and E.coli proteins is similar. The molecular weight of the protein is above 135kD, and the bands of heavy chain 100kD and light chain 50kD hardly exist.
The high imidazole elution of vibrio natrii is further purified by an anion exchange column/SuperDex 200 molecular sieve, and the Abuffer formula of the anion exchange column is as follows: 25mM Tris 8.0,B buffer formula is: 1M NaCl,25mM Tris 8.0. The eluate was conductivity diluted to 7mS/cm, passed through an anion exchange column Source Q, and the Flow Through (FT) was collected, and the elution procedure was linearly followed by a 60% B buffer, with a total elution volume of 200mL. The detection result of the elution UV280nm is shown in FIG. 5, and corresponding samples are collected, protein is prepared, the loading amount of each sample is 5 mu L, and polyacrylamide gel electrophoresis is carried out. The results are shown in FIG. 6. Wherein, the purity of the protein in the tube 7 and the protein in the tube 8 is higher.
7-8 tubes were collected, concentrated to 2mL using 50kD Millipore protein concentrate tube at 2000g speed, loaded onto molecular sieve SuperDex200 with a mobile phase of lysis buffer. The detection result of the elution UV280nm is shown in FIG. 7, corresponding samples are collected, protein is prepared, the loading amount of each sample is 5 mu L, and polyacrylamide gel electrophoresis is carried out. The results are shown in FIG. 6. Wherein the 14 th tube protein is at the peak tip. Tube 14 was collected. The result of SDS-PAGE detection by molecular sieve SUPERDEX200 purification is shown in FIG. 8.
Precast gels of the above samples were all purchased on Biyun day for polyacrylamide gel electrophoresis (SDS-PAGE), and the experimental implementation was performed according to the instructions.
EXAMPLE 6 detection of histidine tag residues of botulinum toxin Using Western-blot
The tube 14 eluted with the molecular sieve of example 5 and the positive control were selected for SDS-PAGE, and the loading buffer was a reduction buffer, purchased from polymeric Mei, M5 5 XSDS-PAGE reducing 5 XScale protein loading buffer, accession number MF145-10. The positive control was performed according to example 4, differing from the sample only in that no TEV cleavage was performed in the middle.
The gel was immunoblotted and transferred to nitrocellulose membrane. The voltage is 25v, the time is 30min, and the transfer film liquid is purchased in the Biyun days. Antibodies were purchased from gold, THE TM His Tag Antibody[HRP]mAb, mouse, were used with reference to their description. After transfer, the developer is purchased from thermo, superSignal TM . As a result, as shown in FIG. 9, the protein was separated into two bands of heavy and light chains by reduction in polyacrylamide gel electrophoresis. Wherein the heavy chain size is between 100kD and 135 kD. The TEV digested sample has no positive strip, and the control group has stronger positive.
EXAMPLE 7 Activity detection of botulinum toxin
The activity of the botulinum toxin prepared in example 5 was tested using a mouse gastrocnemius injection assay, as follows:
the Protein samples obtained in example 5 were diluted to 0.25ng/mL (detection method was ELISA, kit was purchased from Shanghai Chengsheng organism, working curve is shown in FIG. 10, regression equation, abs value is absorbance reading, [ Protein ] means botulinum toxin concentration, R value is greater than 0.99, data quality is reliable), solvent was 0.5%o BSA, 9%o sodium chloride solution. The right hind leg gastrocnemius was injected with 50 μl of botulinum toxin reagent and the left hind leg gastrocnemius was injected with 50 μl of negative control (solvent). As shown in FIG. 11, a protein amount of 5pg could cause paralysis of the right leg of the mice within 12 hours, while the left leg had no effect.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (9)
1. A recombinant botulinum toxin, characterized in that the recombinant botulinum toxin comprises a heavy chain and a light chain linked by disulfide bonds; the amino acid sequence of the heavy chain comprises a protein tag sequence, an ENLYFQ polypeptide sequence and a heavy chain sequence of botulinum toxin from the N end to the C end in sequence,
wherein the 1 st amino acid of the N-terminal of the heavy chain sequence of the botulinum toxin is glycine;
the heavy chain amino acid sequence of the recombinant botulinum toxin is shown as SEQ ID NO.2, and the light chain amino acid sequence is shown as 1 st to 438 th of the sequence shown as SEQ ID NO. 1.
2. A nucleic acid molecule encoding the recombinant botulinum toxin of claim 1.
3. The nucleic acid molecule of claim 2, wherein the nucleic acid molecule encoding a heavy chain is shown in SEQ ID No.3 and/or the nucleic acid molecule encoding a light chain is shown in SEQ ID No. 4.
4. A biological material, characterized in that it comprises the nucleic acid molecule of claim 2 or 3; the biological material is an expression cassette, a vector or a host cell.
5. An engineered bacterium for use in the production of recombinant botulinum toxin, wherein the engineered bacterium comprises the nucleic acid molecule of claim 2 or 3.
6. The engineering bacterium according to claim 5, wherein the engineering bacterium is escherichia coli or vibrio natrii.
7. A method of preparing a botulinum toxin, the method comprising: modifying a host cell such that the host cell expresses the recombinant botulinum toxin of claim 1, culturing the host cell, purifying the recombinant botulinum toxin from the culture using affinity chromatography, and subjecting the purified recombinant botulinum toxin to TEV protease cleavage.
8. The method of claim 7, further comprising the step of sequentially performing anion exchange chromatography and molecular sieve chromatography purification after the TEV protease cleavage.
9. The method of claim 7 or 8, wherein the host cell is vibrio natrii.
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